Proton coupled electron transfer (PCET) underpins primary metabolic steps involving energy transduction, radical initiation and transport and the activation of substrates at cofactors. By examining PCET networks in biomimetic and natural systems, we aim to develop a mechanistic framework in which to understand the structure/function relations of a variety of enzymes and proteins. At a practical level, an understanding of PCET can lead to the development of drugs that directly target reactive radical-based species that cause disease related to oxidative stress including cancer. A major effort will be devoted to the role of PCET in amino acid radical initiation and transport over the 35 [unreadable] electron/proton coupled pathway in E. coli ribonucleotide reductase (RNR). The research plan relies on newly created biochemical and biophysical methods. Radicals will be generated on photoactive peptides or from non-natural amino acids, thereby bypassing the normal radical generation process originating at the di-iron metallocofactor in the R2 subunit of RNR. The competency of these photoinitiated radicals at turning over substrate in the R1 subunit of RNR under various conditions (e.g., radical position along the pathway, variable effector and substrate concentrations) will be established using biochemical probes. New photopeptides will be designed to enable the photochemical intermediates of these "photoRNRs" to be observed and their kinetics for transport measured by transient laser spectroscopy. Non-natural fluorotyrosine amino acids will be exploited to tune the thermodynamics and kinetics of the electron and proton in radical transport by PCET. We will extend studies from the photoRNR R1 subunit and develop photoRNR R2 subunits by introducing photooxidants into the Y356-containing, C-terminal tail of the R2. In tackling the R2 subunit, we will initiate studies to understand the PCET mechanism by which anticancer/antiviral agents can target disease by regulation of RNR. The combination of these steady-state and time-resolved studies should provide the most complete picture to date of PCET in a natural system. The research plan will be extended by investigating the role of PCET in the activation of substrates at Hangman porphyrin constructs, which poise an acid-base functionality over the face of the redox platform. The Hangman construct is a faithful structural and functional model of heme hydroperoxidase enzymes, thus allowing us to examine the PCET mechanism and kinetics of Compound I and II formation. Experiments are presented that allow these kinetics to be measured by stopped-flow and time-resolved spectroscopy. By attaching electron donors and acceptors to the Hangman framework, we will be to examine the mechanism of PCET in which electron and proton transport is bi-directional. This type of transfer is common in biology, but has yet to be captured at a mechanistic level. The principles that emerge from these studies will be applied to explain the functions of a variety of enzymes and proteins that derive their activity from PCET. [unreadable] [unreadable] [unreadable]